The extraction and analysis of formylated phloroglucinol compounds found in eucalypts



The formylated phloroglucinol compounds (FPCs) are perhaps the only group of compounds extracted from eucalypts that influence how much herbivores eat. There are, indeed, many studies that show groups of compounds correlated with feeding but rarely have such compounds been isolated from the plant and tested independently, for example in artificial diets. We now know much about FPCs – distribution in subgenera (Eschler et al. 2000), chemistry (Moore et al. 2004), plant defence (Lawler et al. 2000; Lawler et al. 1998a; Lawler et al. 1998b; Wallis et al. 2002). This protocol describes the rapid extraction and analysis of FPCs.

Whether or not we analyse duplicate samples depends on the sample and the purpose. Some species of eucalypts (Moore et al. 2004) contain a wide variety of FPCs that require a long HPLC separation (80 min) and we usually run single rather than duplicate samples. Other species contain mainly sideroxylonals that we can analyse with much shorter runs (12 min), so sometimes we analyse these in duplicate. If we intend developing prediction equations with near infrared spectroscopy (NIRS) then we invariably analyse these sideroxylonal-rich samples in duplicate. We emphasise, however, that we spent much time analyzing samples in duplicate and documenting the errors at each stage before we relaxed our approach. For example, there is almost no error in repeat HPLC runs on a single vial of sample containing just sideroxylonals or a whole range of FPCs.


The following protocol revolutionized our analysis of FPCs because we replaced a tedious soxhlet extraction and associated drying steps (Wallis et al. 2003) with an extraction using sonication (Wallis and Foley 2005). With the new method we can prepare samples in a few minutes and the number we prepare matches the number we can analyse in a day by HPLC. Thus, if we are analyzing samples for sideroxylonals it is relatively easy to prepare the 120 samples required for 24 h. With the old method we could prepare no more than 7 samples a day because each sample demanded a lot of work and required duplicate extractions. Furthermore, compared to the old method, the new method requires only 1-2% as much sample and a fraction of the solvent. The type of plant tissue is irrelevant; we have analysed leaves, buds, individual flower parts and bark. In many instances we could not have analysed such small samples with the old method. There is, however, one major problem with the assay. All of the compounds we measure are novel and so it is impossible to purchase standards from chemical suppliers. This does not preclude the analysis because it is still possible to express data as “area of a peak per unit dry matter”. Although far from ideal this still allows comparative studies, for instance saying that one individual contains higher concentrations than does another individual.


  • ground tissue samples 60 mg for each sample
  • electronic balance (∓ 0.1 mg)
  • scintillation or similar glass vials (20 mL)
  • racks for 20 mL vials
  • diluent (0.300 g.L{SUP()}-1{SUP} of 2-ethyl-phenol in a solution of 7% water in acetonitrile containing 0.1% TFA)
  • bottle-top dispenser for diluent
  • fumehood
  • ultrasonic bath
  • syringes (2.5 mL)
  • syringe filters (0.22 μm)
  • HPLC vials and caps (1.5 mL)
  • HPLC with photodiode detector or UV detector set at 275 nm and preferably with an autosampler
  • HPLC grade acetonitrile
  • HPLC column (e.g., Wakosil 250 x 4 mm GL 3C18RS; SGE, Ringwood, VIC, Australia)
  • Matching guard-column for HPLC column
  • Milli-Q water
  • High purity trifluoroacetic acid (TFA)
  • Internal standard – 2-ethyl-phenol

Units, terms, definitions
Internal standard – IS



  • Prepare HPLC solvents by adding 0.1% TFA to both HPLC grade acetonitrile and to Milli-Q water. For convenience, we typically prepare 4 L of each by adding 4 mL of TFA (by glass pipette or by mass) to 4 L. Use these solvents both for HPLC and for mixing the extraction diluent. We also have a bottle of pure acetonitrile for washing the column and system at the end of analysis.
  • Prepare 1 L of the extraction diluent with internal standard by weighing exactly 0.3000 g of 2-ethyl-phenol into a small glass beaker. Carefully wash this internal standard into a 1 L volumetric flask using acidified acetonitrile from (1). Place 70 mL of Milli-Q water into the flask and fill to just below the neck with acidified acetonitrile. Stopper and mix well. The reaction is endothermic so top up to the line with acidified acetonitrile when the solution reaches room temperature. Place the HPLC diluent into a dark bottle fitted with an autodispenser set at 5 mL.
  • The sample mass depends on the concentration of FPCs in the starting material. For species that we know contain high concentrations of FPCs (1-10 % of dry matter, DM) we weigh 20 ∓ 0.5 mg of freeze-dried material into a 20 mL glass scintillation vial. For species with much lower concentrations of FPCs we weigh 30 or 50 ∓ 0.5 mg of material.
  • Ensure that the balance is clean and level and that you tare and weigh vessels with the doors closed. Tare a scintillation vial minus the lid. Add the required amount of sample and write down the mass. Tare the vial and sample. Add 5 mL of extraction diluent using the autodispenser. Write down the mass of diluent. Cap the vial. Repeat until you have about 12 vials each containing a known mass of sample and diluent.
  • Place the samples in a rack and suspend in an ultrasonic bath for about 5 min. The time is not critical (Wallis and Foley 2005).
  • Remove the samples from the bath and allow them to settle for 10 min.
  • The final stage of the extraction is filtering the samples directly into HPLC vials. We do this in a fumehood. For this we use one syringe and the same filter for eight samples! I am sure that those with vast resources will frown upon our method but we have tested it thoroughly. For the first sample, simply draw 1.5 mL of sample solution into the syringe and make sure that there is about 0.5-1 mL of air between the plunger and the liquid. Attach a syringe filter to the syringe and filter the sample directly into a labeled HPLC vial. The air pocket ensures that most of the fluid passes the filter.
  • For the second and subsequent samples first draw about 1 mL of sample into the syringe. Extract the plunger to allow this fluid to reach all parts of the syringe. Discard the fluid. Now draw about 2 mL of sample into the syringe, again leaving an air pocket. Attach the filter, press the plunger and discard the first 0.5-1 mL of fluid and then filter the rest into the appropriate vial. The samples are now ready for analysis by HPLC.


It is virtually impossible to describe the operation of an HPLC here. Instead, we provide the running conditions of the HPLC as follows:

We inject 15 μL of sample onto a Wakosil 250 x 4 mm GL 3C18RS (SGE) column maintained at 37oC with a flow rate of 0.75 mL/min on a Waters Alliance Model HPLC. The FPCs are eluted under gradient conditions with 0.1% TFA acid in acetonitrile (A) and 0.1% TFA in water (B) as follows: 60% A/40% B for 5 min, linear gradient to 90% A/10% B at 60 min, hold for 10 min and return to starting conditions over 10 min. For the last analysis we switch to pure acetonitrile after 70 min and then to 100% at 80 min and decrease the flow rate to 0 over 40 min to thoroughly wash the column and instrument.
We measure the peak response at 275nm and quantify the specific FPC compounds present using authentic standards purified in the laboratory.

I might add one note of caution: if you want to start an HPLC quickly then start it slowly. We use a machine shared by many users and assume that it has not been washed appropriately. Thus, we place our column and guard column in the column heater to bring them to operating temperature (40C) while flushing the HPLC. With that done we flush our guard column with the starting solvent and check the back pressure, after which we connect the column and increase the flow rate from 0 to 0.75 mL per minute over 5 minutes. We regularly monitor pressure so that we detect when a column is ageing or requires new frits. Finally, we have not tried to clean our samples more before injection or to use some of the new systems that depend on smaller bore columns and higher pressures but these are both techniques that warrant testing.

Notes and troubleshooting tips

The technique is remarkably simple and hindered only by the inability to purchase standard compounds. Thus, we regularly see compounds that we do not know and often do not have the time or the expertise to identify.
As pointed out above, “if you want to start an HPLC quickly then start it slowly”. We are fastidious with slowly increasing flow rates and with washing the column at the end of a set of analyses. Thus, we have few problems and separation columns last for a long time – about 2000 80 minute analyses.

Links to resources and suppliers
Waters Alliance Model HPLC

Literature references

Eschler BM, Pass DM, Willis R, Foley WJ (2000) Distribution of foliar formylated phloroglucinol derivatives amongst ”Eucalyptus” species. Biochem Syst Ecol 28:813-824

Lawler IR, Foley WJ, Eschler BM (2000) Foliar concentration of a single toxin creates habitat patchiness for a marsupial folivore. Ecology 81:1327-1338

Lawler IR, Foley WJ, Eschler BM, Pass DM, Handasyde K (1998a) Intraspecific variation in ”Eucalyptus” secondary metabolites determines food intake by folivorous marsupials. Oecologia 116:160-169

Lawler IR, Foley WJ, Pass GJ, Eschler BM (1998b) Administration of a 5HT(3) receptor antagonist increases the intake of diets containing ”Eucalyptus” secondary metabolites by marsupials. J Comp Physiol B-Biochem Syst Env Physiol 168:611-618

Moore BD, Wallis IR, Pala-Paul J, Brophy JJ, Willis RH, Foley WJ (2004) Antiherbivore chemistry of ”Eucalyptus” – cues and deterrents for marsupial folivores. J Chem Ecol 30:1743-1769

Wallis IR, Foley WJ (2005) The rapid determination of sideroxylonals in ”Eucalyptus” foliage by extraction with sonication followed by HPLC. Phytochem Anal 16:49-54

Wallis IR, Herlt AJ, Eschler BM, Takasaki M, Foley WJ (2003) Quantification of sideroxylonals in ”Eucalyptus” foliage by HPLC. Phytochem Anal 14:360-365

Wallis IR, Watson ML, Foley WJ (2002) Secondary metabolites in ”Eucalyptus melliodora”: field distribution and laboratory feeding choices by a generalist herbivore, the common brushtail possum. Aust J Zool 50:507-519

[|AOAC – Association of Official Agricultural Chemists] (The AOAC publishes a journal on official methods of chemical analysis)

Health, safety & hazardous waste disposal considerations

The solvents used in the extraction process are dangerous and should be disposed of according to local regulations.


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